Evoscript universal cdna master

The designs of gRNAs were based on the following criteria: targeting Nanog motif hits within Nanog binding sites at the 256-cell stage, and the binding sites are close to the TSS of the first-wave transcribed genes. To exclude the impact of maternal loading mRNAs, we only considered first-wave transcribed genes without maternal loading transcripts. In total, we designed five gRNAs, whose target sites are associated with four genes. For the selected peaks of Nanog binding sites, CRISPR targets were designed at the Nanog motif, and the guide RNAs (gRNAs) were synthesized by PCR amplification and in vitro transcription using the MEGAshortscript T7 kit (Invitrogen AM1354). One nanoliter of Cas9 protein (GenScript Z03389) (4 µM) and gRNAs (150 ng/µL) or Nuclease-free water were injected into zebrafish embryos at the 1-cell stage; 20 embryos developed to desired stages were then collected and removed from their chorions to extract total RNA. The cDNA library was synthesized using EvoScript Universal cDNA Master (Roche 07912374001). Two microliters of cDNA mix were used for RT-qPCR (Roche 4913850001), then relative expression levels of the corresponding Nanog target gene between mutants and water-injected samples were measured by 2^(−ΔΔCt) using actb1 as an internal control gene. All primer sequences for mutation and RT-qPCR are listed in Supplemental Table S6.